Method for the production of L-lysine employing thermophilic corynebacterium thermoaminogenes

ABSTRACT

L-lysine is produced by culturing, in a medium, an L-lysine-producing mutant which belongs to the genus Corynebacterium thermoaminogenes which is capable of growing at 40° C. or higher and which has resistance to S-(2-aminoethyl)-L-cysteine thereby producing and accumulating L-lysine in the culture medium, and collecting L-lysine from the culture medium.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for producing L-lysine by fermentation and to an L-lysine producing mutant.

2. Description of the Background

L-lysine, which is important as an additive for livestock feed, and the like has been mainly prepared by fermentation.

In the industrial preparation of L-lysine by fermentation, some technical factors must be considered in order to achieve economic improvement of the process. These factors include, for example, improvements in yield based on sugar, improvements in concentration of L-lysine which accumulates, shortening of culturing time, and the like.

In culturing, it is important to elevate the temperature for culturing. Culturing is generally carried out at an optimum temperature for L-lysine fermentation. Where conventional L-lysine-producing bacteria are used, the temperature is generally between 28° and 35° C. When culturing begins, the heat of fermentation is generated so that when the system is allowed to stand, the temperature of the culture medium increases with the result that the amount of L-lysine which forms seriously decreases. In order to maintain the temperature of the culture medium in the optimum range, it is necessary to mount a heat exchanger in the fermentation tank and cyclize cool water into the tank. In order to obtain cool water, a cooler must be used. However, since the heat of fermentation generated is very large, the amount of electric energy consumed by the cooler is very large. Accordingly, if the culture temperature for fermentation of L-lysine is increased, the cooling load decreases so that economy of L-lysine production in an industrial scale can be improved.

The method described in Korean Patent Publication No. 85-231 (published August 23, 1985) proposes to increase the culture temperature in the fermentation of L-lysine. That is, the method disclosed employs the L-lysine-producing variant TR-3579 (KFCC 10065) which belongs to the genus Corynebacterium, which has resistance to a lysine analogue and resistance to temperature and further requires homoserine, leucine and valine in combination. Culturing of the variant occurs in a medium at a high temperature (37° to 45° C), thereby producing and accumulating L-lysine in the culture medium.

Since the L-lysine-producing variant used in this method is obtained by improving conventional L-lysine-producing bacteria (grown at 30° to 35° C.) by a mutation which results in the production of L-lysine at a high temperature (37° to 45° C.). the variant has complex auxotrophy of homoserine, leucine and valine as described above, because of the several step mutation employed. Therefore, this method is not practical.

Japanese Patent Application Laid-Open No. 58-170487 discloses a method for producing L-lysine by fermentation which comprises culturing a mutant belonging to the genus Corynebacterium, having a reduced pyruvate kinase acivity, which is capable of producing L-lysine and also which has optional resistance to S-(2-aminoethyl)-L-cysteine in a medium thereby producing and accumulating L-lysine in the culture broth and then collecting L-lysine.

The temperature for fermentation in the method is set at 20° to 40° C., but as is assumed from the only working example (Example 1) in the reference where a temperature of 30° C. is adopted, the growth of the bacteria used is not significant at about 40° C. which is the upper limit of the temperature range for fermentation. Marked formation and accumulation of L-lysine are not observed either. This has been confirmed by experimentation. A need therefore continues to exist for an improved method of producing L-lysine by fermentation.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide bacteria which are capable of producing L-lysine by fermentation at a high temperature which can withstand practical use conditions.

Another object of the present invention is to provide a method for producing L-lysine by fermentation under practical conditions at a high temperature.

Briefly, these objects and other objects of the invention as hereinafter will become apparent can be attained in a method for producing L-lysine by fermentation which comprises culturing, in a medium, an L-lysine-producing mutant belonging to the genus Corynebacterium which is capable of growing even at 40° C. or higher and which has a resistance to S-(2-aminoethyl)-L-cysteine thereby producing and accumulating L-lysine in the culture medium and collecting L-lysine from the culture medium.

The invention also provides an L-lysine-producing mutant per se which can be advantageously utilized in the method described above for producing L-lysine by fermentation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As a result of extensive investigations it has now been found that by using a mutant modified into L-lysine-producing bacteria by imparting AEC resistance to thermophilic bacteria belonging to the genus Corynebacterium which are capable of growing at 40° C. or higher, the objectives described above can be achieved. Based on this finding, the present invention has been accomplished.

The L-lysine-producing mutant of the invention which belongs to the genus Corynebacterium is capable of growing at 40° C. or higher and has AEC resistance. It is obtained by subjecting bacteria belonging to the genus Corynebacterium which are capable of growing at 40° C. or higher, such as for example, L-glutamic acid-producing bacteria, as the parent strain, to a mutation process.

Suitable parent strains include, for example, bacteria belonging to Corynebacterium thermoaminogenes which are described in Japanese Patent Applicant Laid-Open No. 63-240779 and which are isolated from nature.

There is no particular difficulty in collecting the L-lysine-producing mutant after subjecting the parent strain to a mutation process, but a conventionally known method for collecting a chemical-resistant mutant can be used. For example, the parent strain, which has been subjected to a mutation process with N-methyl N'-nitro-N-nitrosoguanidine (250 ug/ml, 30° C., 30 minutes), or the like, is inoculated on an ordinary agar plate medium containing 1 to 2 g/dl of AEC in which the parent strain cannot be grown, and cultured at 40° C. or higher, e.g., 43° C. The resulting colony is collected and the desired mutant obtained.

Examples of the thus collected L-lysine-producing mutant include the following: Corynebacterium thermoaminogenes AJ 12521, Corynebacterium thermoaminogenes AJ 12522, Corynebacterium thermoaminogenes AJ 12523, and Corynebacterium thermoaminogenes AJ 12524. The bacteriological properties of these mutants are shown in Tables 1, 1 through 6.

Corynebacterium thermoaminogenes AJ 12308 and AJ 12309 which are parent strains, have been internationally deposited in the Fermentation Research Institute of the Agency of Industrial Science and Technology of Japan under Accession Nos. FERM BP-1540 and FERM BP-1541, respectively. Furthermore, the mutants Corynebacterium thermoaminogenes AJ 12521, AJ 12522, AJ 12523, and AJ 12524 have also been internationally deposited in the Fermentation Research Institute of the Agency of Industrial Science and Technology of Japan under Accession Nos.FERM BP-3304 , FERM BP-3305, FERM BP-3306 and FERM BP-3307, respectively.

                                      TABLE 1-1                                    __________________________________________________________________________                       Mutant          Parent Strain                                                                          Mutant                                         Parent Strain                                                                          AJ 12521                                                                               AJ 12522                                                                               AJ 12309                                                                               AJ 12523                                                                               AJ 12524                               AJ 12308                                                                               FERM    FERM    FERM    FERM    FERM                                   FERM BP-1540                                                                           BP-3304 BP-3305 BP-1541 BP-3306 BP-3307                      __________________________________________________________________________     Shape:                                                                         (1)                                                                              Shape and                                                                              Rod of 0.7-                                                                            Rod of 0.7-                                                                            Rod of 0.7-                                                                            Rod of 0.7-                                                                            Rod of 0.7-                                                                            Rod of 0.7-                    size of cell                                                                           1.0 × 1.0-4.0                                                                    1.0 × 1.0-4.0                                                                    1.0 × 1.0-4.0                                                                    1.0 × 1.0-4.0                                                                    1.0 × 1.0-4.0                                                                    1.0 × 1.0-4.0                    μm; Cell edges                                                                      μm; Cell edges                                                                      μm; Cell edges                                                                      μm; Cell edges                                                                      μm; Cell edges                                                                      μm; Cell edges                      are rounded.                                                                           are rounded.                                                                           are rounded.                                                                           are rounded.                                                                           are rounded.                                                                           are rounded.                           V-shaped con-                                                                          V-shaped con-                                                                          V-shaped con-                                                                          V-shaped con-                                                                          V-shaped con-                                                                          V-shaped con-                          figuration based                                                                       figuration based                                                                       figuration based                                                                       figuration based                                                                       figuration based                                                                       figuration based                       on snapping                                                                            on snapping                                                                            on snapping                                                                            on snapping                                                                            on snapping                                                                            on snapping                            division is                                                                            division is                                                                            division is                                                                            division is                                                                            division is                                                                            division is                            noted.  noted.  noted.  noted.  noted.  noted.                       (2)                                                                              Polymorphism                                                                           No poly-                                                                               No poly-                                                                               No poly-                                                                               No poly-                                                                               No poly-                                                                               No poly-                               morphism is                                                                            morphism is                                                                            morphism is                                                                            morphism is                                                                            morphism is                                                                            morphism is                            noted but de-                                                                          noted but de-                                                                          noted but de-                                                                          noted but de-                                                                          noted but de-                                                                          noted but de-                          pending on the                                                                         pending on the                                                                         pending on the                                                                         pending on the                                                                         pending on the                                                                         pending on the                         phase of incu-                                                                         phase of incu-                                                                         phase of incu-                                                                         phase of incu-                                                                         phase of incu-                                                                         phase of incu-                         bation, long rod                                                                       bation, long rod                                                                       bation, long rod                                                                       bation, long rod                                                                       bation, long rod                                                                       bation, long rod                       cells, mast cells                                                                      cells, mast cells                                                                      cells, mast cells                                                                      cells, mast cells                                                                      cells, mast cells                                                                      cells, mast cells                      and premature                                                                          and premature                                                                          and premature                                                                          and premature                                                                          and premature                                                                          and premature                          cells are rarely                                                                       cells are rarely                                                                       cells are rarely                                                                       cells are rarely                                                                       cells are rarely                                                                       cells are rarely                       noted.  noted.  noted.  noted.  noted.  noted.                       (3)                                                                              Motility                                                                               None    None    None    None    None    None                         (4)                                                                              Spore   None    None    None    None    None    None                         (5)                                                                              Gram Staining                                                                          Positive                                                                               Positive                                                                               Positive                                                                               Positive                                                                               Positive                                                                               Positive                     (6)                                                                              Antacid Negative                                                                               Negative                                                                               Negative                                                                               Negative                                                                               Negative                                                                               Negative                     __________________________________________________________________________

                                      TABLE 1-2                                    __________________________________________________________________________              Parent Strain                                                                           Mutant            Parent Strain                                                                           Mutant                                     AJ 12308 AJ 12521 AJ 12522 AJ 12309 AJ 12523 AJ                       __________________________________________________________________________                                                           12524                    Physiological                                                                  properties:                                                                    (1)                                                                              Bouillon agar                                                                         Abundant or mod-                                                                        Abundant or mod-                                                                        Abundant or mod-                                                                        Abundant or mod-                                                                        Abundant or                                                                             Abundant or mod-           plate culture                                                                         erate growth;                                                                           erate growth;                                                                           erate growth;                                                                           erate growth;                                                                           erate growth;                                                                           erate growth;                     Colonies are cir-                                                                       Colonies are cir-                                                                       Colonies are cir-                                                                       Colonies are cir-                                                                       Colonies are                                                                            Colonies are cir-                 cular, smooth, en-                                                                      cular, smooth, en-                                                                      cular, smooth, en-                                                                      cular, smooth, en-                                                                      cular, smooth,                                                                          cular, smooth, en-                tire convex,                                                                            tire convex,                                                                            tire convex,                                                                            tire convex,                                                                            tire convex,                                                                            tire convex,                      glossy,  glossy,  glossy,  glossy,  glossy,  glossy,                           opaque or trans-                                                                        opaque or trans-                                                                        opaque or trans-                                                                        opaque or trans-                                                                        opaque or trans-                                                                        opaque or trans-                  lucent, dull                                                                            lucent, dull                                                                            lucent, dull                                                                            lucent, dull                                                                            lucent, dull                                                                            lucent, dull                      yellow butyrous.                                                                        yellow butyrous.                                                                        yellow butyrous.                                                                        yellow butyrous.                                                                        yellow butyrous.                                                                        yellow butyrous.         (2)                                                                              Bouillon agar                                                                         Abundant or mod-                                                                        Abundant or mod-                                                                        Abundant or mod-                                                                        Abundant or mod-                                                                        Abundant or                                                                             Abundant or mod-           slant culture                                                                         erate growth;                                                                           erate growth;                                                                           erate growth;                                                                           erate growth;                                                                           erate growth;                                                                           erate growth;                     filiform, glossy,                                                                       filiform, glossy,                                                                       filiform, glossy,                                                                       filiform, glossy,                                                                       filiform, glossy,                                                                       filiform, glossy,                 dull yellow                                                                             dull yellow                                                                             dull yellow                                                                             dull yellow                                                                             dull yellow                                                                             dull yellow              (3)                                                                              Bouillon                                                                              Moderate growth;                                                                        Moderate growth;                                                                        Moderate growth;                                                                        Moderate growth;                                                                        Moderate growth;                                                                        Moderate growth;           liquid colony becomes                                                                          colony becomes                                                                          colony becomes                                                                          colony becomes                                                                          colony becomes                                                                          colony becomes             culture                                                                               turbid almost                                                                           turbid almost                                                                           turbid almost                                                                           turbid almost                                                                           turbid almost                                                                           turbid almost                     uniformly but                                                                           uniformly but                                                                           uniformly but                                                                           uniformly but                                                                           uniformly but                                                                           uniformly but                     some cells                                                                              some cells                                                                              some cells                                                                              some cells                                                                              some cells                                                                              some cells                        precipitate.                                                                            precipitate.                                                                            precipitate.                                                                            precipitate.                                                                            precipitate.                                                                            precipitate.             (4)                                                                              Bouillon                                                                              Moderate growth;                                                                        Moderate growth;                                                                        Moderate growth;                                                                        Moderate growth;                                                                        Moderate growth;                                                                        Moderate growth;           gelatin stab                                                                          no gelatin is                                                                           no gelatin is                                                                           no gelatin is                                                                           no gelatin is                                                                           no gelatin                                                                              no gelatin is              culture                                                                               liquefied.                                                                              liquefied.                                                                              liquefied.                                                                              liquefied.                                                                              liquefied.                                                                              liquefied.               (5)                                                                              Litmus milk                                                                           Slightly weakly                                                                         Slightly weakly                                                                         Slightly weakly                                                                         Slightly weakly                                                                         Slightly weakly                                                                         Slightly weakly                   alkaline; Neither                                                                       alkaline; Neither                                                                       alkaline; Neither                                                                       alkaline; Neither                                                                       alkaline; Neither                                                                       alkaline; Neither                 liquefaction nor                                                                        liquefaction nor                                                                        liquefaction nor                                                                        liquefaction nor                                                                        liquefaction                                                                            liquefaction nor                  solidification is                                                                       solidification is                                                                       solidification is                                                                       solidification is                                                                       solidification                                                                          solidification is                 noted.   noted.   noted.   noted.   noted.   noted.                   __________________________________________________________________________

                                      TABLE 1-3                                    __________________________________________________________________________                 Parent Strain                                                                          Mutant           Parent Strain                                                                           Mutant                                       AJ 12308                                                                               AJ 12521 AJ 12522                                                                               AJ 12309 AJ 12523                                                                               AJ                       __________________________________________________________________________                                                           12524                    Physiological                                                                  properties:                                                                    (1)                                                                              Reduction of                                                                             Positive                                                                               Positive Positive                                                                               Positive Positive                                                                               Positive                   nitrate                                                                      (2)                                                                              Denitration                                                                              Negative                                                                               Negative Negative                                                                               Negative Negative                                                                               Negative                 (3)                                                                              MR test   Negative or                                                                            Negative or                                                                             Negative or                                                                            Negative or                                                                             Negative or                                                                            Negative or                          slight positive                                                                        slight positive                                                                         slight positive                                                                        slight positive                                                                         slight positive                                                                        slight positive          (4)                                                                              VP test   Positive                                                                               Positive Positive                                                                               Positive Positive                                                                               Positive                 (5)                                                                              Indole formation                                                                         Negative                                                                               Negative Negative                                                                               Negative Negative                                                                               Negative                 (6)                                                                              Formation of                                                                             Positive                                                                               Positive Positive                                                                               Positive Positive                                                                               Positive                   hydrogen sulfide                                                             (7)                                                                              Starch hydrolysis                                                                        Negative                                                                               Negative Negative                                                                               Negative Negative                                                                               Negative                 (8)                                                                              Utilization of                                                                           Does not grow                                                                          Does not grow                                                                           Does not grow                                                                          Does not grow                                                                           Does not grow                                                                          Does not grow              citrate   in Koser me-                                                                           in Koser me-                                                                            in Koser me-                                                                           in Koser me-                                                                            in Koser me-                                                                           in Koser me-                         dium but grows                                                                         dium but grows                                                                          dium but grows                                                                         dium but grows                                                                          dium but grows                                                                         dium but grows                       in Christensen                                                                         in Christensen                                                                          in Christensen                                                                         in Christensen                                                                          in Christensen                                                                         in Christensen                       medium; ren-                                                                           medium; ren-                                                                            medium; ren-                                                                           medium; ren-                                                                            medium; ren-                                                                           medium; ren-                         ders medium                                                                            ders medium                                                                             ders medium                                                                            ders medium                                                                             ders medium                                                                            ders medium                          alkaline.                                                                              alkaline.                                                                               alkaline.                                                                              alkaline.                                                                               alkaline.                                                                              alkaline.                (9)                                                                              Utilization of                                                                           Does not utilize                                                                       Does not utilize                                                                        Does not utilize                                                                       Does not utilize                                                                        Does not utilize                                                                       Does not utilize           inorganic nitrates but util-                                                                     nitrates but util-                                                                      nitrates but util-                                                                     nitrates but util-                                                                      nitrates but                                                                           nitrates but util-         nitrogen  izes ammonium                                                                          izes ammonium                                                                           izes ammonium                                                                          izes ammonium                                                                           izes ammonium                                                                          izes ammonium                        salts.  salts.   salts.  salts.   salts.  salts.                   __________________________________________________________________________

                                      TABLE 1-5                                    __________________________________________________________________________             Parent Strain                                                                         Mutant    Parent Strain                                                                         Mutant                                                 AJ 12308                                                                              AJ 12521                                                                            AJ 12522                                                                            AJ 12309                                                                              AJ 12523                                                                            AJ 12524                                  __________________________________________________________________________     (17) Acid formation                                                            from sugar:                                                                     1 L-Arabinose                                                                         Negative                                                                              Negative                                                                            Negative                                                                            Negative                                                                              Negative                                                                            Negative                                   2 D-Xylose                                                                            Negative                                                                              Negative                                                                            Negative                                                                            Negative                                                                              Negative                                                                            Negative                                   3 D-Glucose                                                                           Positive                                                                              Positive                                                                            Positive                                                                            Positive                                                                              Positive                                                                            Positive                                   4 D-Mannose                                                                           Positive                                                                              Positive                                                                            Positive                                                                            Positive                                                                              Positive                                                                            Positive                                   5 D-Fructose                                                                          Positive                                                                              Positive                                                                            Positive                                                                            Positive                                                                              Positive                                                                            Positive                                   6 D-Galactose                                                                         Negative                                                                              Negative                                                                            Negative                                                                            Negative                                                                              Negative                                                                            Negative                                   7 Maltose                                                                             Positive                                                                              Positive                                                                            Positive                                                                            Positive                                                                              Positive                                                                            Positive                                   8 Sucrose                                                                             Positive                                                                              Positive                                                                            Positive                                                                            Positive                                                                              Positive                                                                            Positive                                   9 Lactose                                                                             Negative                                                                              Negative                                                                            Negative                                                                            Negative                                                                              Negative                                                                            Negative                                  10 Trehalose                                                                           Negative                                                                              Negative                                                                            Negative                                                                            Negative                                                                              Negative                                                                            Negative                                  11 D-Sorbitol                                                                          Negative                                                                              Negative                                                                            Negative                                                                            Negative                                                                              Negative                                                                            Negative                                  12 D-Mannitol                                                                          Negative                                                                              Negative                                                                            Negative                                                                            Negative                                                                              Negative                                                                            Negative                                  13 Inositol                                                                            Negative                                                                              Negative                                                                            Negative                                                                            Negative                                                                              Negative                                                                            Negative                                  14 Glycerine                                                                           Negative                                                                              Negative                                                                            Negative                                                                            Negative                                                                              Negative                                                                            Negative                                  15 Starch                                                                              Negative                                                                              Negative                                                                            Negative                                                                            Negative                                                                              Negative                                                                            Negative                                  __________________________________________________________________________

                                      TABLE 1-6                                    __________________________________________________________________________                  Parent Strain                                                                          Mutant          Parent Strain                                                                           Mutant                                        AJ 12308                                                                               AJ 12521                                                                               AJ 12522                                                                               AJ 12309 AJ 12523                                                                               AJ                       __________________________________________________________________________                                                           12524                    Other                                                                          characterisitcs:                                                               (1)                                                                              Temperature                                                                               By capillary                                                                           By capillary                                                                           By capillary                                                                           By capillary                                                                            By capillary                                                                           By capillary               resistance method in                                                                              method in                                                                              method in                                                                              method in                                                                               method in                                                                              method in                             skimmed milk:                                                                          skimmed milk:                                                                          skimmed milk:                                                                          skimmed milk:                                                                           skimmed milk:                                                                          skimmed milk:                         Alive ab 60° C.,                                                                Alive ab 60° C.,                                                                Alive ab 60° C.,                                                                Alive ab 60° C.,                                                                 Alive ab 60°                                                                    Alive ab 60°                                                            C.,                                   10 mins.                                                                               10 mins.                                                                               10 mins.                                                                               10 mins. 10 mins.                                                                               10 mins.                              Dead ab 65° C.,                                                                 Dead ab 65° C.,                                                                 Dead ab 65° C.,                                                                 Dead ab 65° C.,                                                                  Dead ab 65° C.,                                                                 Dead ab 65°                                                             C.,                                   10 mins.                                                                               10 mins.                                                                               10 mins.                                                                               10 mins. 10 mins.                                                                               10 mins.                 (2)                                                                              Sodium chloride                                                                           Grows in                                                                               Grows in                                                                               Grows in                                                                               Grows in Grows in                                                                               Grows in                   resistance medium con-                                                                            medium con-                                                                            medium con-                                                                            medium con-                                                                             medium con-                                                                            medium con-                           taining 5%                                                                             taining 5%                                                                             taining 5%                                                                             taining 5%                                                                              taining 5%                                                                             taining 5%                            saline. saline. saline. saline.  saline. saline.                  (3)                                                                              Nutrient   Requires biotin                                                                        Requires biotin                                                                        Requires biotin                                                                        Requires biotin                                                                         Requires biotin                                                                        Requires biotin            auxotrophy for growth.                                                                            for growth.                                                                            for growth.                                                                            for growth.                                                                             for growth.                                                                            for growth.              (4)                                                                              Nucleotide 60.2% GC                                                                               60.2% GC                                                                               60.2% GC                                                                               59.5% GC 59.5% GC                                                                               59.5% GC                   composition of                                                                 DNA (Tm method)                                                              (5)                                                                              Dibasic amino                                                                             Meso-   Meso-   Meso-   Meso-    Meso-   Meso-                      acid contained                                                                            diaminopimelic                                                                         diaminopimelic                                                                         diaminopimelic                                                                         diaminopimelic                                                                          diaminopimelic                                                                         diaminopimelic             in cell wall                                                                              acid    acid    acid    acid     acid    acid                     (6)                                                                              Source     Fruit   --      --      Vegetable                                                                               --      --                       __________________________________________________________________________

                                      TABLE 1-4                                    __________________________________________________________________________                 Parent Strain                                                                          Mutant           Parent Strain                                                                           Mutant                                       AJ 12308                                                                               AJ 12521 AJ 12522                                                                               AJ 12309 AJ 12523                                                                               AJ                       __________________________________________________________________________                                                           12524                    (10)                                                                              Pigment  No pigment is                                                                          No pigment is                                                                           No pigment is                                                                          No pigment is                                                                           No pigment                                                                             No pigment is               formation                                                                               formed extra-                                                                          formed extra-                                                                           formed extra-                                                                          formed extra-                                                                           formed extra-                                                                          formed extra-                        cellularly.                                                                            cellularly.                                                                             cellularly.                                                                            cellularly.                                                                             cellularly.                                                                            cellularly.              (11)                                                                              Urease test                                                                             Negative or                                                                            Negative or                                                                             Negative or                                                                            Negative or                                                                             Negative or                                                                            Negative or                          slightly positive                                                                      slightly positive                                                                       slightly positive                                                                      slightly positive                                                                       slightly positive                                                                      slightly positive        (12)                                                                              Oxidase  Negative                                                                               Negative Negative                                                                               Negative Negative                                                                               Negative                 (13)                                                                              Catalase Positive                                                                               Positive Positive                                                                               Positive Positive                                                                               Positive                 (14)                                                                              Growth range                                                                            Grows well at                                                                          Grows well at                                                                           Grows well at                                                                          Grows well at                                                                           Grows well                                                                             Grows well at                        Ph 7-8.5;                                                                              Ph 7-8.5;                                                                               Ph 7-8.5;                                                                              Ph 7-8.5;                                                                               Ph 7-8.5;                                                                              Ph 7-8.5;                            grows well at                                                                          grows well at                                                                           grows well at                                                                          grows well at                                                                           grows well                                                                             grows well at                        35-45° C.;                                                                      35-45° C.;                                                                       35-45° C.;                                                                      35-45° C.;                                                                       35-45° C.;                                                                      35-45° C.;                    grows slightly                                                                         grows slightly                                                                          grows slightly                                                                         grows slightly                                                                          grows slightly                                                                         grows slightly                       at 46-50° C.                                                                    at 46-50° C.                                                                     at 46-50° C.                                                                    at 46-50° C.                                                                     at 46-50° C.                                                                    at 46-50°                                                               C.                       (15)                                                                              Behavior to                                                                             Aerobic or                                                                             Aerobic or                                                                              Aerobic or                                                                             Aerobic or                                                                              Aerobic or                                                                             Aerobic or                  oxygen   facultative                                                                            facultative                                                                             facultative                                                                            facultative                                                                             facultative                                                                            facultative                          anerobic                                                                               anerobic anerobic                                                                               anerobic anerobic                                                                               anerobic                 (16)                                                                              O-F test Grows fer-                                                                             Grows fer-                                                                              Grows fer-                                                                             Grows fer-                                                                              Grows fer-                                                                             Grows fer-                  (glucose)                                                                               mentatively to                                                                         mentatively to                                                                          mentatively to                                                                         mentatively to                                                                          mentatively                                                                            mentatively to                       form acid.                                                                             form acid.                                                                              form acid.                                                                             form acid.                                                                              form acid.                                                                             form                     __________________________________________________________________________                                                           acid.               

The medium used for producing and accumulating L-lysine using the present L-lysine-producing mutant, may be any conventional known medium for producing L-lysine. Conventional media containing carbon sources, nitrogen sources, inorganic salts, and, if necessary, other organic trace nutrients which the microorganism uses, can be used. Any carbon source may be used so long as the mutant used can use the same. Examples of the carbon sources include glucose, sucrose, maltose, starch hydrolysates containing them, sugars such as molasses, and the like; alcohols such as ethanol, propanol and the like; organic acids such as acetic acid, propionic acid, and the like. In addition, normal paraffin, and the like may also be used singly or in combination with other carbon sources, depending on the strain. As nitrogen sources, inorganic and organic nitrogen sources may be used including ammonium salts such as ammonium acetate, ammonium chloride, ammonium phosphate, and the like., nitrates, urea, ammonia, and furthermore, meat extract, and the like. Suitable inorganic salts include KH₂ PO₄, MgSO₄, FeSO₄, MnSO₄ and the like which are ordinarily used. Suitable organic trace nutrients include vitamins, fatty acids and nucleic acids, and peptone, casamino acid, yeast extract or protein hydrolysate containing them.

Incubation is preferably carried out under aerobic conditions. By carrying out shake culture or aerial spinner culture for 2 to 4 days, and adjusting the pH of the medium to a range from 5 to 9, preferably 7 to 8.5 at the growth temperature of the bacteria used, i.e., about 25° to about 50° C., preferably about 35° to about 45° C. in order to achieve high L-lysine productivity, L-lysine can be produced and accumulated in the culture medium in a marked quantity.

Conventional methods for collecting L-lysine from the culture medium can be used. For example, L-lysine may be recovered by the conventional ion exchange resin method, crystallization, or the like in an appropriate combination.

As described above, the temperature for fermentation of L-lysine in accordance with the present invention is at the growth temperature of bacteria used, namely about 25° to about 50° C., preferably 35° to 45° C. at which L-lysine productivity increases. Examples 2 and 3 actually demonstrate that L-lysine was formed and accumulated in a remarkable quantity, by regulating the fermentation temperature at 43° C. on a laboratory scale.

Based on the experimental results, the cooling load for removing heat generated by fermentation on a commercial scale was calculated by trial. In the event that fermentation of L-lysine was performed using a fermentation tank having a volume of, e.g., 200 kiloliters, the cooling load for maintaining the optimum fermentation temperature, e.g., 30° C., for L-lysine-producing bacteria was 30 millionkilocalories per batch, whereas in the present invention the cooling load for maintaining the fermentation temperature not exceeding 43° C. was 3 million kilocalories per batch, when the L-lysine-producing mutant of the present invention was used. As is clear from the above data, the cooling load can be reduced by 90% by the method of the present invention.

Having now generally described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.

EXAMPLE 1 (harvest of mutant)

After Corynebacterium thermoaminogenes AJ 12308 was treated with N-methyl-N-nitro-N-nitrosoguanidine (250 μg/ml, 30° C., 30 minutes) in a conventional manner, incubation was conducted at 43° C. in a medium obtained by supplementing the minimum medium below with 1.5 g/dl of AEC. Colonies grown at 43° C. in the medium were harvested.

    ______________________________________                                         Composition of minimum medium:                                                 ______________________________________                                         Glucose          2.0          g/dl                                             Urea             0.25         g/dl                                             Ammonium sulfate 1.0          g/dl                                             KH.sub.2 PO.sub.4                                                                               0.1          g/dl                                             MgSO.sub.4.7H.sub.2 O                                                                           0.04         g/dl                                             FeSO.sub.4.7H.sub.2 O                                                                           1.0          mg/dl                                            L-Alanine        50           mg/dl                                            Nicotinamide     0.5          mg/dl                                            MnSO.sub.4.4H.sub.2 O                                                                           1.0          mg/dl                                            Biotin           5.0          μg/dl                                         Thiamine hydrochloride                                                                          10           μg/dl                                         NaCl             5            mg/dl                                            pH 7.2                                                                         ______________________________________                                    

Among the mutants thus obtained, AJ 12521 and AJ 12522 were harvested as mutants having excellent L-lysine productivity.

In a similar manner, AJ 12523 and AJ 12524 were harvested using Corynebacterium thermoaminogenes AJ 12309 as the parent strain.

The four mutants thus obtained were previously grown on glucose-bouillon agar medium at 43° C. One platinum loop aliquotos were inoculated respectively on 20 ml of sterile medium charged into a shaking flask of 500 ml volume and having the following composition.

    ______________________________________                                         Composition of medium:                                                         ______________________________________                                         Beat molasses       10         g/dl                                            Ammonium sulfate    5          g/dl                                            KH.sub.2 PO.sub.4   0.1        g/dl                                            MgSO.sub.4.7H.sub.2 O                                                                              40         mg/dl                                           Biotin              50         μg/dl                                        Calcium carbonate (separately                                                                      5          g/dl                                            sterilized and added)                                                          pH 7.0                                                                         ______________________________________                                    

These mutants were cultured at 43° C. for 72 hours. Lysine was accumulated as shown in Table 2.

                  TABLE 2                                                          ______________________________________                                         Strain   Concentration of L-lysine accumulated (g/dl)                          ______________________________________                                         AJ 12521 3.2                                                                   AJ 12522 2.9                                                                   AJ 12523 2.8                                                                   AJ 12524 3.0                                                                   ______________________________________                                    

EXAMPLE 2

In a shaking 500 ml volume flask, 20 ml of aqueous medium having the following composition was separately charged followed by sterilization at 110° C. for 10 minutes with steam.

    ______________________________________                                         Composition of medium:                                                         ______________________________________                                         Glucose          10           g/dl                                             Ammonium sulfate 4.5          g/dl                                             KH.sub.2 PO.sub.4                                                                               0.1          g/dl                                             MgSO.sub.4.7H.sub.2 O                                                                           0.04         g/dl                                             FeSO.sub.4.7H.sub.2 O                                                                           1.0          mg/dl                                            MnSO.sub.4.4H.sub.2 O                                                                           1.0          mg/dl                                            Biotin           5.0          μg/dl                                         Thiamine hydrochloride                                                                          20           μg/dl                                         Concentrate of soybean                                                                          1.5          ml/dl                                            protein hydrolysate                                                            with hydrochloric acid                                                         (total nitrogen, 7%)                                                           Calcium carbonate                                                                               5            g/ml                                             (separately sterilized                                                         and added)                                                                     pH 7.0                                                                         ______________________________________                                    

One platinum loop of Corynebacterium thermoaminogenes AJ 12521 which had been previously grown on a glucose bouillon slant was inoculated on the thus prepared medium charged in a flask. Shaking culture was conducted at 43° C. for 72 hours.

The amount of L-lysine produced in the medium after culturing for 72 hours was colorimetrically determined using the ninhydrin reaction. The results indicate that L-lysine was produced in an amount of 3.0 g/dl.

Furthermore, solutions corresponding to 50 flasks cultured in a similar manner which completed incubation were collected and the cells and calcium salt were removed by centrifugation. About 1 liter of the thus obtained supernatant was passed through a strongly acidic ion exchange resin (AMBERLITE" IR-120 (OH type)) to adsorb L-lysine thereonto. The adsorbed L-lysine was eluted with 3% ammonia water and the eluate was concentrated under reduced pressure. After hydrochloric acid was added to the concentrate, the mixture was cooled and L-lysine was precipitated as L-lysine hydrochloride dihydrate to give 26.5 g of crystals.

EXAMPLE 3

One platinum loop each of Corynebacterium thermoaminogenes AJ 12523 and the parent strain AJ 12309 Was taken from the slant and inoculated on 50 ml of the aqueous seed culture medium described below. Aerial spinner culturing was performed at 43° C. for 18 hours to prepare a seed culture medium.

    ______________________________________                                         Composition of seed culture medium:                                            ______________________________________                                         Glucose          1.5          g/dl                                             Ammonium sulfate 0.3          g/dl                                             Urea             0.1          g/dl                                             KH.sub.2 PO.sub.4                                                                               0.1          g/dl                                             MgSO.sub.4.7H.sub.2 O                                                                           0.04         g/dl                                             FeSO.sub.4.7H.sub.2 O                                                                           1.0          mg/dl                                            MnSO.sub.4.4H.sub.2 O                                                                           1.0          mg/dl                                            Biotin           5.0          μg/dl                                         Thiamine hydrochloride                                                                          20           μg/dl                                         Concentrate of soybean                                                                          2.0          ml/dl                                            protein hydrolysate                                                            with hydrochloric acid                                                         (total nitrogen, 7%)                                                           pH 7.5                                                                         ______________________________________                                    

On the other hand, 300 ml aliquots of aqueous medium for main fermentation having the composition described below were each charged into a glass-made jar fermenter of 1 liter volume and sterilized in a conventional manner.

Each medium was inoculated with 15 ml of the seed culture medium and aerial spinner culturing was initiated at 43° C.

    ______________________________________                                         Composition of medium for main fermentation:                                   ______________________________________                                         Glucose          2.0          g/dl                                             Ammonium sulfate 0.5          g/dl                                             Urea             0.2          g/dl                                             KH.sub.2 PO.sub.4                                                                               0.1          g/dl                                             MgSO.sub.4.7H.sub.2 O                                                                           0.04         g/dl                                             FeSO.sub.4.7H.sub.2 O                                                                           1.0          mg/dl                                            MnSO.sub.4.4H.sub.2 O                                                                           1.0          mg/dl                                            Biotin           5.0          μg/dl                                         Thiamine hydrochloride                                                                          50           μg/dl                                         Nicotinamide     1.0          mg/dl                                            Concentrate of soybean                                                                          3.0          ml/dl                                            protein hydrolysate                                                            with hydrochloric acid                                                         (total nitrogen, 7%)                                                           pH 7.2                                                                         ______________________________________                                    

A mixture of acetic acid and ammonium acetate (molar ratio of acetic acid:ammonium acetate in the mixture was 1:0.25; concentration of acetic acid in the mixture was 60%) was added to the culture medium to maintain the pH of the medium between 7.2 and 8.0. Incubation was performed at 43° C. for 55 hours.

The results are shown in Table 3.

                  TABLE 3                                                          ______________________________________                                                      Amount of L-Lysine                                                Strain Used  Accumulated (g/dl)                                                ______________________________________                                         AJ 12523     3.2                                                               AJ 12309     0.03                                                              (parent)                                                                       ______________________________________                                    

From 300 ml of the solution obtained after completion of fermentation of AJ 12323, 1.0 g of L-lysine hydrochloride dihydrate was obtained as crystals in a manner similar to Example 2.

EXAMPLE 4

In a shaking flask of 500 ml volume, 20 ml of the same medium as used in Example 2 was charged followed by sterilization at 110° C. for 10 minutes with steam. One platinum loop of Corynebacterium thermoaminogenes AJ 12524, which had been previously grown on glucose-bouillon slant, was inoculated on the medium followed by shaking culturing at 43° C. for 50 hours.

After 0.2 ml of the culture medium was charged into a glass-made jar fermenter, 5 ml of cooled physiological saline was added thereto. After stirring, the mixture was centrifuged at 4500 rpm for 10 minutes and the supernatant was discharged.

A mixture of 1.5 ml of 0.2 M phosphate buffer (pH 7.5) and 1.5 ml of the reaction solution described below was added and the mixture was stirred. The cells were suspended followed by a shaking reaction (120 rpm) at 43° C. for 2 hours.

    ______________________________________                                         Reaction solution:                                                             ______________________________________                                         Glucose        1             g/dl                                              (NH.sub.4).sub.2 SO.sub.4                                                                     0.4           g/dl                                              MgSO.sub.4.7H.sub.2 O                                                                         0.04          g/dl                                              Biotin         500           μg/dl                                          pH 7.5                                                                         ______________________________________                                    

Thereafter, centrifugation (4500 rpm, 10 minutes) was carried out and the concentration of L-lysine in the supernatant was analyzed by liquid chromatography. The results showed that 0.2 g/dl of L-lysine had accumulated.

The present invention provides a method for producing L-lysine on a industrial scale in which the cooling load for eliminating heat generated upon fermentation is reduced. Further, the method requires no amino acid nutrients and can be practically used.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein. 

What is claimed as new and desired to be secured by Letters Patent of the United States is:
 1. A method for producing L-lysine by fermentation comprising:culturing in a nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic salts a strain of Corynebacterium thermoaminogenes which is capable of growing at 40° C. or higher and is resistant to S-(2-aminoethyl)-L-cysteine under conditions effective for the production of L-lysine wherein said strain is selected from the group consisting of Corynebacterium thermoaminogenes AJ 12521, FERM BP-3304, Corynebacterium thermoaminogenes AJ 12522, FERM BP-3305, Corynebacterium thermoaminogenes AJ 12523, FERM BP-3306, Corynebacterium thermoaminogenes AJ 12524, FERM BP-3307; and collecting L-lysine from the culture medium.
 2. The method of claim 1, wherein culturing is conducted at a temperature of 25° to about 50° C.
 3. The method of claim 2, wherein the culturing temperature ranges from 35° to 45° C.
 4. The method of claim 1, wherein culturing is conducted at a pH of 5 to
 9. 5. The method of claim 4, wherein culturing is conducted at a pH of 7 to 8.5. 